Vibrational Circular Dichroism Measurements Research Articles

Vibrational circular dichroism of carbohydrate films formed from aqueous solutions

Vibrational circular dichroism (VCD) spectra in the entire 2000–900 cm −1 region have been recorded, for the first time, for films of carbohydrates prepared from aqueous solutions. Eight different carbohydrates, α- d-glucopyranosyl-(1→4)- d-glucose, cyclomaltohexaose, α- d-glucopyranosyl α- d-glucopyranoside, β- d-glucopyranosyl-(1→6)- d-glucose, β- d-glucopyranosyl-(1→4)- d-glucose, d-glucose, and both enantiomers of 6-deoxygalactose and of allose, were investigated. The VCD spectra obtained for films are found to be identical to the corresponding spectra obtained for aqueous solutions of carbohydrates. These measurements demonstrate several advantages of significant importance. The strong infrared absorption of water has prevented, in the past, the pursuit for routine applications of VCD in determining the structures of carbohydrates in aqueous solutions. This limitation is not present for film studies because water solvent is removed in the process of preparing the films. Also, strong infrared absorption of water at 1650 cm −1 requires the use of very short-pathlength (6 μm) cells for measurements on aqueous solutions. This requirement and concomitant inconveniences (such as laborious assembling of a demountable liquid cell or purchasing an expensive variable pathlength liquid cell) have been eliminated for film measurements. The removal of interfering water absorption in film studies resulted in higher light throughput and better signal-to-noise ratios for VCD measurements. Another point of significance is that the amount of carbohydrate sample required for VCD measurements on films is approximately one to two orders of magnitude smaller than that required for corresponding VCD measurements on aqueous solutions. Since carbohydrate samples can now be studied as films, VCD spectroscopy becomes much more broadly applicable for carbohydrates than previously believed. The present work, in combination with other film measurements in our laboratory, indicate that VCD studies on films can be used more generally, providing a convenient and powerful approach for probing structural information for biologically important compounds.

Vibrational circular dichroism of protein films.

Vibrational circular dichroism (VCD) spectra in the 1800-1400 cm(-)(1) region have been measured for the first time for protein films prepared from aqueous buffer solutions. These measurements demonstrate several advantages of significant importance. First, the interference from infrared absorption of water in the amide I region, which is a serious limitation for measurements in water solutions, is eliminated. Second, the amounts of protein samples required for VCD measurements on films are approximately 2 orders of magnitude smaller than those required for the same in water solutions. In addition, the amide I absorption and VCD bands of protein films are found to be independent of film orientation. Furthermore, characteristic VCD patterns have been observed for protein films whose secondary structure is dominated by alpha-helix, beta-sheet, and alpha + beta combinations. These results demonstrate that VCD can be used to study the structure of proteins in the film state.

Absolute configuration determination of chiral molecules in the solution state using vibrational circular dichroism.

Advances in the measurement, calculation, and application of vibrational circular dichroism (VCD) for the determination of absolute configuration are described. The purpose of the review is to provide an up-to-date perspective on the capability of VCD to solve problems of absolute stereochemistry for chiral molecules primarily in the solution state. The scope of the article covers the experimental methods needed for the accurate measurement of VCD spectra and the theoretical steps required to systematically deduce absolute configuration. Determination of absolute configuration of a molecule by VCD requires knowledge of its conformation or conformational distribution, and hence VCD analysis necessarily provides solution-state conformation information, in many cases available by no other method, as an additional benefit. Comparisons of the advantages and limitations of VCD relative to other available chiroptical methods of analysis are also presented.

Vibrational Circular Dichroism Spectrum of 1-Amino-2-Propanol

Vibrational circular dichroism (VCD) spectra of 1-amino-2-propanol, of neat sample and in dilute solution of CDCl3, were measured in 2000 to 800 cm(-1) region. Two VCD bands, which appear at 1272 and 1412 cm(-1) in dilute solution, were assigned, respectively, to the OH bending mode and the mode that is influenced by the OH bending of the most stable conformer. The assignments were confirmed according to the prediction by a high-level density function theory (DFT) calculation and the isotope shift of deuterated samples. The joint analysis of the spectra obtained by the VCD and ordinary infrared (IR) measurements seems to be a useful way of using vibrational spectroscopy to investigate the hydrogen bonding system in solution.

Theoretical simulation of a polarization modulator based on mechanical rotation of a polarizing element

The properties of three alternative designs for a polarization modulator of potential use for the measurement of vibrational circular dichroism (VCD) are evaluated and compared by use of Mueller calculus. The analysis shows that the combination of a fixed polarizer plus either a photoelastic modulator or a rotating quarter-wave plate possesses nearly the same capability for generation of time-varying, circularly polarized light. However, a modulator composed of a rotating polarizer plus a fixed birefringent plate entails considerable theoretical and experimental difficulties for use in the measurement of VCD spectra. While VCD spectra obtained with the rotating devices can be calibrated in the same manner as spectra obtained with a photoelastic modulator, Mueller analysis shows that the form of the resultant calibration signal will have a different shape. The relevant expressions for VCD and linear dichroism as well as the calibration signals are presented, and consequences for practical realization of these experiments are discussed.

Measurement of Vibrational Circular Dichroism below ∼600 cm−1: Progress towards Meeting the Challenge

The measurement of vibrational circular dichroism (VCD) below ∼600 cm−1 is one of the experimental challenges of the current times. Polarization-division interferometry is considered to be the most efficient approach for realizing these measurements. A crucial component for successful applications of this interferometry is the beamsplitter, which divides the incoming beam according to polarization. Free-standing metal wires and the metal wires deposited on a mylar substrate have been employed as beamsplitters, and their performance has been evaluated for the ∼700–10 cm−1 region. VCD measurements obtained for the enantiomers of α-pinene in the 400–250 cm−1 region are presented. The current status and future developments needed for routine far-infrared VCD measurements are summarized.

Measurement of Dispersive Vibrational Circular Dichroism: Signal Optimization and Artifact Reduction

Artifacts, or spurious signals, are common problems in the measurement of optical activity. Until recently they have limited the applicability of vibrational circular dichroism (VCD) in cases of weak ellipticity—for example, in structural studies of small peptides. The artifacts in dispersive VCD to be discussed here can be from the optical and electronic hardware as well as from the data processing methods in the software. The data acquisition and processing methods reported here reduce system artifacts in VCD measurements, particularly for samples with weak ellipticity.

Observations on the Measurement of Vibrational Circular Dichroism with Rapid-Scan and Step-Scan FT-IR Techniques

Extensive tests to determine an optimal method for using a Bio-Rad FTS-60A spectrometer for measurement of vibrational circular dichroism (VCD) in both step-scan and rapid-scan modes are reported. In the latter case, results of “fast-scan” and “slow-scan” experiments are also presented for comparison. In step-scan mode, phase modulation (PM) during the polarization-modulation measurements is found to be detrimental to the signal-to-noise ratio (S/N), but is very useful for the normal IR measurements at slow step speeds. VCD S/N is improved by doing sequential spectral collection of the polarization-modulated spectrum without PM and the instrument response spectra with phase modulation. Systematic comparative studies are used to identify optimal parameter sets for doing the step-scan VCD measurements. These conditions use more rapid data collection with faster steps than previously suggested, which permits a more realistic evaluation of S/N and reproducibility of the spectra through comparison of spectra collected in blocks. Example VCD spectra are presented for small molecules in nonaqueous solution measured in short times and for proteins in aqueous solution measured over longer times scales. Near-IR FT-VCD measured in step-scan mode at both moderate resolution and high S/N are presented. At this stage, fast-scan, slow-scan, and step-scan techniques are shown to be virtually equivalent in the mid-IR, but the reduction in frequency dependence of the phase correction and the new capability for VCD measurement in the near-IR favor continued development of step-scan techniques.

New Design for Fourier Transform Infrared Vibrational Circular Dichroism Spectrometers

Fourier transform infrared (FT-IR) spectrometers are commonly designed with small-area detectors and tight focusing mirrors. Vibrational circular dichroism (VCD) measurements made with such FT-IR instruments contain polarization artifacts, and VCD measurements on both enantiomers (or one enantiomer and racemic mixture) are required in order to reduce these artifacts. This restriction limits the VCD measurements to only those samples for which both enantiomers (or one enantiomer and racemic mixture) are available. Recently a modified design was reported in the literature where the mirrors between sample and detector were replaced with a BaF2 lens, and a larger-area detector was substituted for the smaller-area counterpart. These modifications successfully alleviated some of the artifact problems. This design, however, is not suitable for polarizing interferometers, where polarizations exiting the interferometer are to be preserved to a high degree of purity. In addition, it is not clear whether the throughput enhancement advantage realized with a larger-area detector completely offsets the disadvantage from increased noise with detector area. Furthermore, BaF2 lenses reduce the broad range routinely available on an FT-IR instrument. Here we report a new design that replaces all the mirrors at the exit port of the interferometer with two KBr lenses and retains the full spectral range (4000–400 cm−1) of mid-infrared FT-IR spectrometers. VCD measurements obtained with small- (1 × 1 mm) and large- (4 × 4 mm) area detectors are found to have similar signal quality.

Vibrational circular dichroism measurements of ligand vibrations in haem and non-haem metalloenzymes

We have measured the vibrational circular dichroism (VCD) spectra of the stretching vibrations of azide and cyanide ligated to the Fe3+ atoms of haemoglobin (Hb) and myoglobin (Mb). The antisymmetric azide-stretch of the low-spin haems have an anomalously large g-value of ca. -1 x 10(-3). In contrast, CN- has a g-value of ca. +2.4 x 10(-3). We also show, for the first time, that a significant VCD occurs for the azide ligand antisymmetric stretches of non-haem proteins; we measure a g-value of ca. -1 x 10(-4) for azide bound to haemerythrin. We have examined the mechanism of the VCD phenomenon by: (1) reconstituting Mb with haems substituted such that they insert differently in the haem pocket; (2) replacing the Fe3+ with Mn3+; (3) examining proteins where replacements occur for E-7 His and E-11 Val distal amino acids close to the haem and (4) examining an Mb mutant where the proximal F-8 His is replaced by Gly, and where an imidazole ligand inserts into the resulting crevice and binds to the haem in a way similar to that of the proximal histidine in the native protein. The VCD anisotropy appears insensitive to the haem substituent replacements used in this study. Exchange of the E-7 distal His or the E-11 Val has a dramatic effect on the g-value. Exchange of the F-8 proximal His reverses the sign of the g-value for the azide complex, but not for the cyanide complex. The work to date indicates that VCD has the potential to become a sensitive technique for examining the structure of metalloenzymes. Work is needed to determine the mechanism giving rise to the large g-values and to correlate the VCD spectrum with the metalloenzyme structure at the active site.

A Method for the Determination of the Zero-Path-Difference Position in a Fourier Transform Infrared Spectrometer: Application to Magnetic Vibrational Circular Dichroism

Some modern FT-IR spectrometers, without a white light source, use a pre-scan of the signal to determine the zero path difference for purposes of scan initialization. The interferograms in vibrational circular dichroism (VCD) and other low signal-to-noise ratio experiments have little or no centerburst, which can cause problems. This can lead to wasted data collections and to instabilities between successively collected blocks of scans. In this modification, a PbSe detector is used to detect frequencies above 2200 cm−1 extracted from a small fraction of the beam. This near-IR signal is then added to the demodulated differential signal to enable the instrument to initialize scans. Since, in this example, the VCD spectra are typically obtained below 2000 cm−1, there is no spectral interference between the two detector signals. However, use of two signals from different detectors can lead to phase problems, which have been solved with the use of polarization calibration methods. As an example, construction of such a modified spectrometer for magnetic VCD measurement is described in detail. At 0.5 cm−1 resolution, these modulated spectra evidence resolution of the rotational structure in the bands. Such data were unobservable with the unmodified instrument.

Vibrational Circular Dichroism Measurement in the Frequency Range of 800 to 650 cm−1

The development of dispersive instrumentation for the measurement of vibrational circular dichroism (VCD) with a lower frequency limit of ∼650 cm−1 is reported. VCD spectra of 3-methylcyclohexanone, α-pinene, and 3-bromocamphor in the frequency range of 800 to 650 cm−1 are presented to illustrate instrumental performance. The spectra obtained are superior to earlier spectra obtained with the use of a Fourier transform spectrometer.

Vibrational Circular Dichroism Measurements by Optical Subtraction FT-IR Spectrometry

The principles of dual-beam FT-IR vibrational circular dichroism (VCD) spectrometry are described. Results from this technique are presented and compared to the results from double modulation FT-IR spectrometry. Performance of dual-beam FT-IR VCD measurements are evaluated for signal-to-noise ratio and optical alignment. The use of Fresnel rhombs for mid-infrared circular dichroism spectrometry is described for the first time.

Polarization Demodulation: A New Approach to the Reduction of Polarization Artifacts from Vibrational Circular Dichroism Spectra

In vibrational circular dichroism (VCD) measurements and other polarization modulation techniques, artifact signals often arise due to polarization sensitivity of the optical components located subsequent to the sample. In this paper, we describe a method for demodulating the light beam after passage through the sample in such a manner that only one linearly polarized state is allowed to reach the detector, thus eliminating these artifacts. The method is a beam reversal technique in which the light subsequent to the sample is redirected through the photoelastic modulator in such a way that the retardation produced on the first traversal through the modulator is removed. The light is returned to its original state of linear polarization prior to detection. The method is applicable to Fourier transform as well as to dispersive spectrometers since demodulation is effected for all wavelengths simultaneously. The results of preliminary demodulation experiments are presented and found to be more than 90% efficient in removing circular dichroism intensity of a birefringent origin.